The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene (in a foreign DNA) at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps, which include extensive genetic characterization, breeding, and evaluation in field trials, eventually leading to the selection of an elite event.
The unequivocal identification of an elite event is becoming increasingly important in view of discussions on Novel Food/Feed, segregation of GMO and non-GMO products and the identification of proprietary material. Ideally, such identification method is both quick and simple, without the need for an extensive laboratory set-up. Furthermore, the method should provide results that allow unequivocal determination of the elite event without expert interpretation, but which hold up under expert scrutiny if necessary.
GAT-ZM1 was selected as an elite event in the development of corn resistant to the herbicide Liberty(copyright), by transformation of corn with plasmid pUC/Ac comprising the pat gene encoding tolerance to phosphinothricin. It is commercially sold as. Liberty Link(copyright) maize, such as, for instance, Liberty Link(copyright) A6460LL sold by AgriGold/Akin Seed Company. The tools for use in simple and unequivocal methods for identification elite event GAT-ZM1 in biological samples are described herein.
The present invention relates to methods for identifying elite event GAT-ZM1 in biological samples, which methods are based on primers or probes which specifically recognize the 5xe2x80x2 and/or 3xe2x80x2 flanking sequence of GAT-ZM 1.
More specifically, the invention relates to a method comprising of amplifying a sequence of a nucleic acid present in biological samples, using a polymerase chain reaction with at least two primers, one of which recognizes the 5xe2x80x2 or 3xe2x80x2 flanking region of GAT-ZM1, the other which recognizes a sequence within the foreign DNA, to obtain a DNA fragment of between 100 and 350 bp. Preferably, the primers recognize a sequence within the 5xe2x80x2 flanking region of GAT-ZM1, most preferably within the 5xe2x80x2 flanking region of SEQ ID No. 6, and a sequence within the foreign DNA, respectively. Especially preferably, the primer recognizing the 5xe2x80x2 flanking region comprises the nucleotide sequence of SEQ ID No 11 and the primer recognizing a sequence within the foreign DNA comprises the nucleotide sequence of SEQ ID No 12 described herein.
The present invention more specifically relates to a method for identifying elite event GAT-ZM1 in biological samples, which method comprises amplifying a sequence of a nucleic acid present in a biological sample, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No 11 and SEQ ID No 12 respectively, to obtain a DNA fragment of between 180 and 220 bp, preferably of about 200 bp.
The present invention further relates to the specific flanking sequences of GAT-ZM1 described herein, which can be used to develop specific identification methods for GAT-ZM1 in biological samples. More particularly, the invention relates to the 5xe2x80x2 and or 3xe2x80x2 flanking regions of GAT-ZM1 which can be used for the development of specific primers and probes. The invention further relates to identification methods for the presence of GAT-ZM1 in biological samples based on the use of such specific primers or probes.
The invention further relates to kits for identifying elite event GAT-ZM1 in biological samples, said kits comprising at least one primer or probe which specifically recognizes the 5xe2x80x2 or 3xe2x80x2 flanking region of GAT-ZM1.
Preferably the kit of the invention comprises, in addition to a primer which specifically recognizes the 5xe2x80x2 or 3xe2x80x2 flanking region of GAT-ZM1, a second primer which specifically recognizes a sequence within the foreign DNA of GAT-ZM1, for use in a PCR identification protocol. Preferably, the kit of the invention comprises two specific primers, one of which recognizes a sequence within the 5xe2x80x2 flanking region of GAT-ZM1, most preferably within the 5xe2x80x2 flanking region of SEQ ID No. 6, and the other which recognizes a sequence within the foreign DNA. Especially preferably, the primer recognizing the 5xe2x80x2 flanking region comprises the nucleotide sequence of SEQ ID No 11 and the primer recognizing the transgene comprises the nucleotide sequence of SEQ ID No 12 described herein.
The invention firther relates to a kit for identifying elite event GAT-ZM1 in biological samples, said kit comprising the PCR primers having the nucleotide sequence of SEQ ID No. 11 and SEQ ID No. 12 for use in the GAT-ZM1 PCR identification protocol described herein.
The invention also relates to a kit for identifying elite event GAT-ZM1 in biological samples, which kit comprises a specific probe having a sequence which corresponds (or is complementary to) a sequence having between 80% and 100% sequence identity with a specific region of GAT-ZM1 . Preferably the sequence of the probe corresponds to a specific region comprising part of the 5xe2x80x2 or 3xe2x80x2 flanking region of GAT-ZM1. Most preferably the specific probe has (or is complementary to) a sequence having between 80% and 100% sequence identity to the sequence between nucleotide 286 and 466 of SEQ ID No. 6.
The methods and kits encompassed by the present invention can be used for different purposes such as, but not limited to the following: to identify GAT-ZM1 in plants, plant material or in products such as, but not limited to food or feed products (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality (i.e. percentage pure material) of plant material comprising GAT-ZM1.
The invention further relates to the 5xe2x80x2 and/or 3xe2x80x2 flanking regions of GAT-ZM1 as well as to the specific primers and probes developed from the 5xe2x80x2 and/or 3xe2x80x2 flanking sequences of GAT-ZM1.
The incorporation of a recombinant DNA molecule in the plant genome typically results from transformation of a cell or tissue (or from another genetic manipulation). The particular site of incorporation is either due to xe2x80x9crandomxe2x80x9d integration or is at a predetermined location (if a process of targeted integration is used).
The DNA introduced into the plant genome as a result of transformation of a plant cell or tissue with a recombinant DNA or xe2x80x9ctransforming DNAxe2x80x9d is hereinafter referred to as xe2x80x9cforeign DNAxe2x80x9d comprising one or more xe2x80x9ctransgenesxe2x80x9d. Thus, foreign DNA may comprise both recombinant DNA as well as newly introduced, rearranged DNA of the plant. However, the term xe2x80x9cplant DNAxe2x80x9d in the context of the present invention will refer to DNA of the plant which is found in the same genetic locus in the corresponding wild-type plant. The foreign DNA can be characterized by the location and the configuration at the site of incorporation of the recombinant DNA molecule in the plant genome. The site in the plant genome where a recombinant DNA has been inserted is also referred to as the xe2x80x9cinsertion sitexe2x80x9d or xe2x80x9ctarget sitexe2x80x9d. Insertion of the recombinant DNA into the plant genome can be associated with a deletion of plant DNA, referred to as xe2x80x9ctarget site deletionxe2x80x9d. A xe2x80x9cflanking regionxe2x80x9d or xe2x80x9cflanking sequencexe2x80x9d as used herein refers to a sequence of at least 20 bp, preferably at least 50 bp, and up to 5000 bp of the plant genome which is located either immediately upstream of and contiguous with or immediately downstream of and contiguous with the foreign DNA. Transformation procedures leading to random integration of the foreign DNA will result in transformants with different flanking regions, which are characteristic and unique for each transformant. When the recombinant DNA is introduced into a plant through traditional crossing, its insertion site in the plant genome, or its flanking regions will generally not be changed. An xe2x80x9cinsertion regionxe2x80x9d as used herein refers to the region corresponding to the region of at least 40 bp, preferably at least 100 bp, and up to 10000 bp, encompassed by the sequence which comprises the upstream and/or the downstream flanking region of a foreign DNA in the plant genome. Taking into consideration minor differences due to mutations within a species, an insertion region will retain, upon crossing into a plant of the same species, at least 85%, preferably 90%, more preferably 95%, and most preferably 100% sequence identity with the sequence comprising the upstream and downstream flanking regions of the foreign DNA in the plant originally obtained from transformation.
An event is defined as a (artificial) genetic locus that, as a result of genetic manipulation, carries a transgene comprising at least one copy of a gene of interest. The typical allelic states of an event are the presence or absence of the foreign DNA. An event is characterized phenotypically by the expression of the transgene. At the genetic level, an event is part of the genetic makeup of a plant. At the molecular level, an event can be characterized by the restriction map (e.g. as determined by Southern blotting), by the upstream and/or downstream flanking sequences of the transgene, the location of molecular markers and/or the molecular configuration of the transgene. Usually transformation of a plant with a transforming DNA comprising at least one gene of interest leads to a multitude of events, each of which is unique.
An elite event, as used herein, is an event which is selected from a group of events, obtained by transformation with the same transforming DNA or by back-crossing with plants obtained by such transformation, based on the expression and stability of the transgene(s) and its compatibility with optimal agronomic characteristics of the plant comprising it. Thus the criteria for elite event selection are one or more, preferably two or more, advantageously all of the following:
a) That the presence of the foreign DNA does not compromise other desired characteristics of the plant, such as those relating to agronomic performance or commercial value;
b) That the event is characterized by a well defined molecular configuration which is stably inherited and for which appropriate tools for identity control can be developed;
c) That the gene(s) of interest show(s) a correct, appropriate and stable spatial and temporal phenotypic expression, both in heterozygous (or hemizygous) and homozygous condition of the event, at a commercially acceptable level in a range of environmental conditions in which the plants carrying the event are likely to be exposed in normal agronomic use.
It is preferred that the foreign DNA is associated with a position in the plant genome that allows easy introgression into desired commercial genetic backgrounds.
The status of an event as an elite event is confirmed by introgression of the elite event in different relevant genetic backgrounds and observing compliance with one, two or all of the criteria e.g. a), b) and c) above.
An xe2x80x9celite eventxe2x80x9d thus refers to a genetic locus comprising a foreign DNA, which answers to the above-described criteria. A plant, plant material or progeny such as seeds can comprise one or more elite events in its genome.
The tools developed to identify an elite event or the plant, plant material comprising an elite event, or products which comprise plant material comprising the elite event are based on the specific genomic characteristics of the elite event, such as, a specific restriction map of the genomic region comprising the foreign DNA, molecular markers or the sequence of the flanking region(s) of the foreign DNA.
Once one or both of the flanking regions of the foreign DNA have been sequenced, primers and probes can be developed which specifically recognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of a sample by way of a molecular biological technique. For instance a PCR method can be developed to identify the elite event in biological samples (such as samples of plants, plant material or products comprising plant material). Such a PCR is based on at least two xe2x80x9cspecific primersxe2x80x9d preferably one recognizing a sequence within the 5xe2x80x2 or 3xe2x80x2 flanking region of the elite event and the other recognizing a sequence within the foreign DNA. The primers preferably have a sequence of between 15 and 35 nucleotides which under optimized PCR conditions xe2x80x9cspecifically recognizexe2x80x9d a sequence within the 5xe2x80x2 or 3xe2x80x2 flanking region of the elite event and the foreign DNA of the elite event respectively, so that a specific fragment (xe2x80x9cintegration fragmentxe2x80x9d) is amplified from a nucleic acid sample comprising the elite event. This means that only the targeted integration fragment, and no other sequence in the plant genome or foreign DNA, is amplified under optimized PCR conditions.
Preferably, the integration fragment has a length of between 50 and 500 nucleotides, most preferably of between 100 and 350 nucleotides. Preferably the specific primers have a sequence which is between 80 and 100% identical to a sequence within the 5xe2x80x2 or 3xe2x80x2 flanking region of the elite event and the foreign DNA of the elite event, respectively, provided the mismatches still allow specific identification of the elite event with these primers under optimized PCR conditions. The range of allowable mismatches however, can easily be determined experimentally and are known to a person skilled in the art.
As the sequence of the primers and their relative location in the genome are unique for the elite event, amplification of the integration fragment will occur only in biological samples comprising (the nucleic acid of) the elite event. Preferably when performing a PCR to identify the presence of GAT-ZM1 in unknown samples, a control is included of a set of primers with which a fragment within a xe2x80x9chousekeeping genexe2x80x9d of the plant species of the event can be amplified. Housekeeping genes are genes that are expressed in most cell types and which are concerned with basic metabolic activities common to all cells. Preferably, the fragment amplified from the housekeeping gene is a fragment which is larger than the amplified integration fragment. Depending on the samples to be analyzed, other controls can be included.
Standard PCR protocols are described in the art, such as in xe2x80x9cPCR Applications Manualxe2x80x9d (Roche Molecular Biochemicals, 2nd Edition, 1999). The optimal conditions for the PCR, including the sequence of the specific primers, is specified in a xe2x80x9cPCR identification protocolxe2x80x9d for each elite event. It is however understood that a number of parameters in the PCR identification protocol may need to be adjusted to specific laboratory conditions, and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA may require adjustment of, for instance, the amount of primers, polymerase and annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR identification protocol. These adjustments will however be apparent to a person skilled in the art, and are furthermore detailed in current PCR application manuals such as the one cited above.
Alternatively, specific primers can be used to amplify an integration fragment that can be used as a xe2x80x9cspecific probexe2x80x9d for identifying GAT-ZM1 in biological samples. Contacting nucleic acid of a biological sample, with the probe, under conditions which allow hybridization of the probe with its corresponding fragment in the nucleic acid, results in the formation of a nucleic acid/probe hybrid. The formation of this hybrid can be detected (e.g. labeling of the nucleic acid or probe), whereby the formation of this hybrid indicates the presence of GAT-ZM1. Such identification methods based on hybridization with a specific probe (either on a solid phase carrier or in solution) have been described in the art. The specific probe is preferably a sequence which, under optimized conditions, hybridizes specifically to a region within the 5xe2x80x2 or 3xe2x80x2 flanking region of the elite event and preferably also comprising part of the foreign DNA contiguous therewith (hereinafter referred to as xe2x80x9cspecific regionxe2x80x9d). Preferably, the specific probe comprises a sequence of between 50 and 500 bp, preferably of 100 to 350 bp which is at least 80%, preferably between 80 and 85%, more preferably between 85 and 90%, especially preferably between 90 and 95%, most preferably between 95% and 100% identical (or complementary) to the nucleotide sequence of a specific region. Preferably, the specific probe will comprise a sequence of about 15 to about 100 contiguous nucleotides identical (or complementary) to a specific region of the elite event.
A xe2x80x9ckitxe2x80x9d as used herein refers to a set of reagents for the purpose of performing the method of the invention, more particularly, the identification of the elite event GAT-ZM1 in biological samples. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers, as described above. Optionally, the kit can further comprise any other reagent described herein in the PCR identification protocol. Alternatively, according to another embodiment of this invention, the kit can comprise a specific probe, as described above, which specifically hybridizes with a specific region in the DNA of GAT-ZM1 in biological samples, to identify the presence of GAT-ZM1 nucleic acid therein. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer, label) for identification of GAT-ZM1 in biological samples, using the specific probe.
The kit of the invention can be used, and its components can be specifically adjusted, for purposes of quality control (e.g., purity of seed lots), detection of the elite event in plant material or material comprising or derived from plant material, such as but not limited to food or feed products.
As used herein, xe2x80x9csequence identityxe2x80x9d with regard to nucleotide sequences (DNA or RNA), refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences. The alignment of the two nucleotide sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window-size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can, e.g., be conveniently performed using the programs of the IntelligeneticsTM Suite (Intelligenetics Inc., CA) or the sequence analysis software package of the Genetics Computer Group (GCG, University of Wisconsin Biotechnology center). Sequences are indicated as xe2x80x9cessentially similarxe2x80x9d when such sequences have a sequence identity of at least about 75%, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%. It is clear than when RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. xe2x80x9cComplementary toxe2x80x9d as used herein refers to the complementarity between the A and T (U), and G and C nucleotides in nucleotide sequences.
The term xe2x80x9cprimerxe2x80x9d as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 basepairs, but longer sequences can be employed. Primers may be provided in double-stranded form, though the single-stranded form is preferred. Probes can be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.
The term xe2x80x9crecognizingxe2x80x9d as used herein when referring to specific primers, refers to the fact that the specific primers specifically hybridize to a nucleic acid sequence in the elite event under the conditions set forth in the method (such as the conditions of the PCR identification protocol), whereby the specificity is determined by the presence of positive and negative controls.
The term xe2x80x9chybridizingxe2x80x9d as used herein when referring to specific probes, refers to the fact that the probe binds to a specific region in the nucleic acid sequence of the elite event under standard stringency conditions. Standard stringency conditions as used herein refers to the condition for hybridization described herein or to the conventional hybridizing conditions as described by Sambrook et al. (1989) (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY) which for instance can comprise the following steps: 1) immobilizing plant genomic DNA fragments on a filter, 2) prehybridizing the filter for 1 to 2 hours at 42xc2x0 C. in 50% formamide, 5xc3x97SSPE, 2xc3x97Denhardt""s reagent and 0.1% SDS, or for 1 to 2 hours at 68xc2x0 C. in 6xc3x97SSC, 2xc3x97Denhardt""s reagent and 0.1% SDS, 3) adding the hybridization probe which has been labeled, 4) incubating for 16 to 24 hours, 5) washing the filter for 20 min. at room temperature in 1xc3x97SSC, 0.1% SDS, 6) washing the filter three times for 20 min. each at 68xc2x0 C. in 0.2xc3x97SSC, 0.1% SDS, and 7) exposing the filter for 24 to 48 hours to X-ray film at xe2x88x9270xc2x0 C. with an intensifying screen.
As used in herein, a biological sample is a sample of a plant, plant material or products comprising plant material. The term xe2x80x9cplantxe2x80x9d is intended to encompass corn (Zea mays) plant tissues, at any stage of maturity, as well as any cells, tissues, or organs taken from or derived from any such plant, including without limitation, any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts. xe2x80x9cPlant materialxe2x80x9d, as used herein refers to material which is obtained or derived from a plant. Products comprising plant material relate to food, feed or other products which are produced using plant material or can be contaminated by plant material. It is understood that, in the context of the present invention, such biological samples are tested for the presence of nucleic acids specific for GAT-ZM1, implying the presence of nucleic acids in the samples. Thus, the methods referred to herein for identifying elite event GAT-ZM1 in biological samples, relate to the identification in biological samples of nucleic acids which comprise the elite event.
As used herein xe2x80x9ccomprisingxe2x80x9d is to be interpreted as specifying the presence of the stated features, integers, steps, reagents or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA sequence which is functionally or structurally defined, may comprise additional DNA sequences, etc.
The following examples describe the development of tools for the identification of elite event GAT-ZM1 in biological samples.
Unless otherwise stated, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.
In the description and examples, reference is made to the following sequences: